Figure 1

Preparation and evaluation of canine chimeric anti-PD-L1 mAb c4G12. (a) Schematic image of rat mAb and canine chimeric mAb. (b) Expression and purification of c4G12. CHO-DG44 cell lines which stably produce c4G12 were established and the culture supernatant was purified by protein A derivative. SDS-PAGE and Coomassie brilliant blue staining were performed and the images were analysed by densitometry to evaluate the protein purities. Left panel, reduced condition; Right panel, non-reduced condition. Rat mAb 4G12 was used as a control protein. Full-length gels are presented in Supplementary Figure S4. (c) Blocking of PD-1/PD-L1 binding by c4G12. cPD-1-Ig was coated on a microwell plate and binding of cPD-L1-Ig, which had been preincubated with various concentrations of anti-PD-L1 mAbs 4G12 or c4G12, was detected on the plate. Rat IgG and dog IgG were used as control antibodies. (d) Blocking of CD80/PD-L1 binding by c4G12. cCD80-Ig was coated on a plate and cPD-L1-Ig binding was evaluated as described above. Each point represents a mean value of relative OD (%) obtained from three independent experiments. Error bar; SE. Statistical analysis was performed by Tukey’s test. *p < 0.05; n.s., not significant.